Vasoconstriction, blood cell adhesion, and inflammation are each potentially devastating events in sickle cell disease (SCD). Sickle red cells (SS RBCs) demonstrate complex membrane and biologic abnormalities. Nitric oxide (NO) delivered by RBCs is both critical in the maintenance of vasodilation and a potent anti-inflammatory agent. ATP is also released by RBCs and signals increases in blood flow to meet O2 demand, typically by enhancing NO synthesis. While normal (AA) RBCs act as a hypoxia sensor by releasing both ATP and bioactive nitric oxide (NO), leading to NO-dependent vasodilation, SS RBCs are deficient in both content and ability to release both NO and ATP. The ability of SS RBCs to adhere to the endothelium and to activate leukocytes as well as other cells, along with their failure to induce pulmonary vasodilation, may result in part from their deficiencies in membrane-bound bioactive S-nitrosothiol (SNO) and ATP. Our preliminary data show that loading SS RBCs with NO/SNO down-regulates SS RBC adhesion, S RBC-stimulated leukocyte adhesion, and vaso-occlusion in vivo and modulates the vasoconstrictive pulmonary phenotype. Inhibition of ATP release by RBCs also induces RBC adhesion and pulmonary vasoconstriction. In SCD, abnormal vascular tone, cell adhesion, leukocyte activation, and inflammation are all believed to contribute to the pathophysiology of vaso-occlusion, which is central to both painful crises and acute and chronic organ damage. Thus, our central hypothesis is that NO and ATP deficits in SS RBCs directly contribute to both SS RBC adhesion, S RBC-induced activation of leukocytes, and pulmonary vasoconstriction. We further postulate that restoration of NO and ATP content in either SS or transfused (stored) AA RBCs will also improve some of the measurable adverse effects of SS RBCs in the lung. To test our hypothesis and progress toward achieving improved therapies for SCD, we have combined the efforts of investigators with expertise in SS RBC biology, transfusion medicine, and pulmonary physiology in order to 1) Determine the influence of NO/SNO- and ATP-repletion on pulmonary hemodynamics and gas exchange in isolated lungs and intact mice transfused with SS RBCs alone or in combination with strategies that modulate RBC adhesive events; 2) Test whether NO and ATP repletion of SS RBCs or stored AA RBCs can relieve vaso-occlusion in a mouse model in vivo; and 3) Determine the effect of SS RBC NO and ATP on the activation of RBC adhesion receptors in vitro, the signaling pathways involved, and RBC activation of leukocytes. Our long-term goal is to improve vascular tone, cell adhesion, and cell activation in SCD by identifying remediable SS RBC abnormalities and thereby reduce vaso-occlusion. This work will allow development of new therapeutic approaches to prevent and control vaso-occlusion and tissue damage in SCD. PUBLIC HEALTH RELEVANCE: Relevance Sickle cell disease (SCD) affects approximately 100,000 individuals in the USA and several million worldwide. Development of new therapies to treat or prevent vaso-occlusion, inflammation and abnormal lung function in SCD is critical to reducing the disease's morbidity and mortality. We propose to test the hypothesis, supported by our preliminary data, that the nitric oxide and ATP deficiencies of sickle red blood cells contribute to the abnormal red cell adhesion, cell-cell interactions, inflammatory leukocyte activation, vaso-occlusion, and adverse pulmonary hemodynamic changes caused by sickle red cells in a variety of model systems.